A detailed depiction of the 'integrin adhesome', consisting of a complex network of 156 components linked together and modified by 690 interactions is presented. Different views of the network reveal several functional 'subnets' that are involved in switching on or off many of the molecular interactions within the network, consequently affecting cell adhesion, migration and cytoskeletal organization. Examination of the adhesome network motifs reveals a relatively small number of key motifs, dominated by three-component complexes in which a scaffolding molecule recruits both a signalling molecule and its downstream target. We discuss the role of the different network modules in regulating the structural and signalling functions of cell-matrix adhesions. Top-down and bottom-up approaches for studying the integrin adhesomeCell-extracellular matrix (ECM) interactions are mediated through specialized subcellular sites that contain specific adhesion receptors, cytoskeletal elements and a wide variety of interconnecting adaptor proteins [1][2][3] . These adhesion complexes permit cells to sense multiple extracellular signals that specify the chemical identity, geometry and physical properties of the ECM 4,5 . Thus, cells behave differently on two-and three-dimensional matrices 6 , distinguish between different ECM components 7 , can detect differences in adhesive ligand density 8 , and respond to mechanical perturbation and surface rigidity 9,10 . Competing Financial Interests:The authors declare no competing financial interests.Website -www.adhesome.org contains the adhesome database of components and interactions with an interface that allows dynamical navigation between hyperlinked subnets created for each component and for many network motifs within the adhesome network.Publisher's Disclaimer: Disclaimer: Nature Publishing Group has a collaboration with the Cell Migration Consortium for the creation and maintenance of the Cell Migration gateway (http://www.cellmigration.org/), but has no role in generating or curating the Cell Migration Consortium database content. As always, Nature Cell Biology Editors have been fully independent and solely responsible for the editorial content and peer review of this Analysis article. NIH Public Access Author ManuscriptNat Cell Biol. Author manuscript; available in PMC 2009 August 31. Published in final edited form as:Nat Cell Biol. 2007 August ; 9(8): 858-867. doi:10.1038/ncb0807-858. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptTo understand the mechanisms underlying these diverse responses, in-depth characterization of individual proteins or pathways 11,12 , and collection of information about multiple components that concertedly form the presumed adhesome 13,14 , have been undertaken. Each of these approaches has limitations, and individually is unlikely to explain how the adhesion machinery senses environmental cues and responds to them. However, combining data from the two approaches could produce new mechanistic insights into the structu...

Cellular locomotion is driven by repeated cycles of protrusion of the leading edge, formation of new matrix adhesions and retraction of the trailing edge. In this study we addressed the molecular composition and dynamics of focal complexes, formed under the leading lamellae of motile cells, and their maturation into focal adhesions. We combined phase-contrast and fluorescence microscopy approaches to monitor the incorporation of phosphotyrosine and nine different focal adhesion proteins into focal complexes in endothelial cells, migrating into an in vitro `wound'. We show that newly formed complexes are located posterior to an actin-, VASP- and α-actinin-rich region in the lammelipodium. They are highly tyrosine phosphorylated, contain β3-integrin, talin, paxillin and low levels of vinculin and FAK, but are apparently devoid of zyxin and tensin. The recruitment of these proteins into focal complexes occurs sequentially, so that their specific protein composition depends on their age. Interestingly, double color, time-lapse movies visualizing both paxillin and zyxin, indicated that the transition from paxillin-rich focal complexes to definitive, zyxin-containing focal adhesions, takes place only after the leading edge stops advancing or retracts. These observations illuminate, for the first time, early stages in focal complex assembly and the dynamic process associated with its transformation into focal adhesion.

Diverse cellular processes are carried out by distinct integrin-mediated adhesions. Cell spreading and migration are driven by focal complexes; robust adhesion to the extracellular matrix by focal adhesions; and matrix remodeling by fibrillar adhesions. The mechanism(s) regulating the spatio-temporal distribution and dynamics of the three types of adhesion are unknown. Here, we combine live-cell imaging, labeling with phosphospecific-antibodies and overexpression of a novel tyrosine phosphomimetic mutant of paxillin, to demonstrate that the modulation of tyrosine phosphorylation of paxillin regulates both the assembly and turnover of adhesion sites. Moreover, phosphorylated paxillin enhanced lamellipodial protrusions, whereas non-phosphorylated paxillin was essential for fibrillar adhesion formation and for fibronectin fibrillogenesis. We further show that focal adhesion kinase preferentially interacted with the tyrosine phosphomimetic paxillin and its recruitment is implicated in high turnover of focal complexes and translocation of focal adhesions. We created a mathematical model that recapitulates the salient features of the measured dynamics, and conclude that tyrosine phosphorylation of the adaptor protein paxillin functions as a major switch, regulating the adhesive phenotype of cells.

The adhesion of cells to the extracellular matrix is a dynamic process, mediated by a series of cell-surface and matrix-associated molecules that interact with each other in a spatially and temporally regulated manner. These interactions play a major role in tissue formation, cellular migration and the induction of adhesion-mediated transmembrane signals. In this paper, we show that the formation of matrix adhesions is a hierarchical process, consisting of several sequential molecular events. One of the earliest steps in surface recognition is mediated, in some cells, by a 1 microm-thick cell-surface hyaluronan coat, which precedes the establishment of stable, cytoskeleton-associated adhesions. The earliest forms of these integrin-mediated contacts are dot-shaped FXs (focal complexes), which are formed under the protrusive lamellipodium of migrating cells. These adhesions recruit, sequentially, different anchor proteins that are involved in binding the actin cytoskeleton to the membrane. Conspicuous in its absence from FXs is zyxin, which is recruited to these sites only on retraction of the leading edge and the transformation of the FXs into a focal adhesion. Continuing application of force to focal adhesions results in the formation of fibrillar adhesions and reorganization of the extracellular matrix. The formation of these adhesions depends on actomyosin contractility and matrix pliability.

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E-cadherin is the major adhesion receptor in epithelial adherens junctions, which connect cells to form tissues and are essential for morphogenesis and homeostasis. The mechanism by which E-cadherin monomers cluster and become organized in adherens junctions remains poorly understood. Here, using superresolution microscopy techniques in combination with structure-informed functional mutations, we found that loosely organized clusters of approximately five E-cadherin molecules that form independently of cis or trans interactions, and that are delimited by the cortical F-actin meshwork, are the precursors of trans-ligated adhesive clusters that make up the adherens junction. The density of E-cadherin clusters was wide ranged, and notably, we could detect densities consistent with the crystal lattice structure at the core of adhesive clusters, which were dependent on extracellular domain interactions. Thus, our results elucidate the nanoscale architecture of adherens junctions, as well as the molecular mechanisms driving its assembly.

Although myosin II filaments are known to exist in non-muscle cells, their dynamics and organization are incompletely understood. Here, we combined structured illumination microscopy with pharmacological and genetic perturbations, to study the process of actomyosin cytoskeleton self-organization into arcs and stress fibres. A striking feature of the myosin II filament organization was their 'registered' alignment into stacks, spanning up to several micrometres in the direction orthogonal to the parallel actin bundles. While turnover of individual myosin II filaments was fast (characteristic half-life time 60 s) and independent of actin filament turnover, the process of stack formation lasted a longer time (in the range of several minutes) and required myosin II contractility, as well as actin filament assembly/disassembly and crosslinking (dependent on formin Fmnl3, cofilin1 and α-actinin-4). Furthermore, myosin filament stack formation involved long-range movements of individual myosin filaments towards each other suggesting the existence of attractive forces between myosin II filaments. These forces, possibly transmitted via mechanical deformations of the intervening actin filament network, may in turn remodel the actomyosin cytoskeleton and drive its self-organization.

E-cadherin-mediated cell-cell adhesion and signaling plays an essential role in development and maintenance of healthy epithelial tissues. Adhesiveness is conferred by cadherin extracellular domains, and is regulated by an assembly of adaptors and enzymes associated with the cytoplasmic tail. Here, we employed proximity biotinylation and quantitative proteomics to isolate and identify 612 proteins in the vicinity of E-cadherin's cytoplasmic tail. We used a structureinformed database of protein-protein interactions to construct the most comprehensive E-cadherin interactome to date, containing 89 known E-cadhesome components and 346 novel proteins. Moreover, through cloning and expression of GFP-tagged fusion proteins we localized 26 of the novel proteins to adherens junctions. Finally, employing calcium depletion and myosin inhibition we show the E-cadherin interactome to be remarkably robust to perturbation and essentially independent of cell-cell junctions or actomyosin contractility.